Principal Investigator/Program Director (Last, first, middle): Sorescu, Dan RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? l Yes m No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes l No IACUC Approval Date: 03-22-2006 Animal Welfare Assurance Number A-3180-01 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 4322-projectabstract.pdf Mime Type: application/pdf 7. * Project Narrative 2366-projectnarrative.pdf Mime Type: application/pdf 8. Bibliography &References Cited 8340-Bibliographyandreferencesfinal.pdfMime Type: application/pdf 9. Facilities &Other Resources 5053-Resources.pdf Mime Type: application/pdf 10. Equipment 2357-Equipment.pdf Mime Type: application/pdf Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Sorescu, Dan NAD(P)H oxidases (Nox) are major sources of ROS in cardiac fibroblasts by reversibly oxidizing the cysteine residues in target proteins. We recently published that deletion of the isoform Nox4 in murine cardiac fibroblasts prevent Angiotensin II and TGF-1-induced cardiac fibrosis in vitro. The intimate mechanisms by which Nox4-released ROS mediate the pro-fibrotic transcriptional response to Ang II and TGF-1 are unknown. In this proposal we present additional data which supports that 1) Nox4 mediates TGF-1 and Ang II upregulation of fibrosis markers (SM- actin, collagen I, fibronectin, and CTGF) by controlling activation of transcription factors Smad2/3;2) Nox4 binds to and regulates the small GTPase RhoA which in turns controls activation and nuclear translocation of transcription factors Smad2/3;3) the novel redox-sensitive transcription factor FoxO3A is required for TGF-1 and Ang II expression of pro-fibrotic proteins and finally 4) Nox4 regulates de-acetylation of FoxO3A which is required for its proper function. Deletion of Nox4, Foxo3A, Smad2/3 prevent expression of SM- actin and Smad2/3 form a transcriptional complex with FoxO3A. Our central hypothesis is that Nox4-released ROS are essential for Ang II and TGF-1-induced fibrosis by inhibiting the tyrosine-phosphatase Shp2 that negatively regulates the activation and nuclear cooperation of transcription factors Smad2/3 and FoxO3A. The following specific aims will be accomplished: Aim 1. Establish the mechanism by which Nox4 controls activation of RhoA-Smad2/3 pathway and fibrotic proteins expression in response to Ang II and TGF-1 in murine cardiac fibroblasts. We will test whether Nox4 inhibits Shp2 and stimulates RhoA activation, required for Smad2/3 activation and nuclear translocation. Aim 2. Determine the specific mechanism by which Nox4 modulates FOXO3A- dependent SM- actin induction in response to Ang II and TGF-1. We plan to test that Nox4 regulates the transcriptional activity of Foxo3A by regulating activation of the redox-sensitive c-Jun-N-kinase (JNK) via inhibition of Shp2 and this is essential for induction of pro-fibrotic phenotype in response to Ang II and TGF-1. Aim 3. Establish the role of Nox4-based oxidase in cardiac fibrosis and hypertrophy in response to Ang II in mice in which Nox4 is over-expressed or deleted. We will test whether Nox4 is essential in vivo in Ang II-induced cardiac fibrosis by using Nox4 knockout or Nox4 overexpressing mice. Project Description Page 6